Esterases, DNA encoding therefor and vectors and host incorporating same

ABSTRACT

A novel DNA is provided which encodes an enzyme having esterolytic activity isolated from Aspergillus. Also provided for is a method of isolating DNA encoding an enzyme having esterolytic activity from organisms which possess such DNA, transformation of the DNA into a suitable host organism, expression of the transformed DNA and the use of the expressed esterase protein in feed as a supplement, in textiles for the finishing of such textiles prior to sale, in starch processing or production of foods such as baked bread.

This application is a continuation of application Ser. No. 08/722,713filed Sep. 30, 1996, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is directed to a novel esterolytic enzyme, novelgenetic material encoding that enzyme and esterolytic proteins developedtherefrom. In particular, the present invention provides an esterasederived from Aspergillus, a DNA encoding that esterase, vectorscomprising that DNA, host cells transformed with that DNA and a proteinproduct produced by such host cells.

Xylan, next to cellulose, is the most abundant renewable polysaccharidein nature. It is the major hemicellulosic component in plants and islocated predominantly in the secondary cell walls of angiosperms andgymnosperms. The composition and structure of xylan are more complicatedthan that of cellulose and can vary quantitatively and qualitatively invarious woody plant species, grasses, and cereals. Xylan is aheteropolymer in which the constituents are linked together not only byglycosidic linkages but also by ester linkages. Ferulic acid is the mostabundant hydroxycinnamic acid found in plants and is known to beesterified to arabinose in wheat bran, wheat flour, barley straw, maize,sugar-cane bagasse, rice straw and other monocotyledons and also foundesterified to galactose residues in pectins of sugar beet, spinach andother dicotyledons. p-Coumaric acid is also linked in a similar fashionin monocots. The presence of these phenolic acids has been shown tolimit cell-wall biodegradation and play significant roles in cell wallextension and stabilization through cross-linking heteroxylan chains byforming phenolic dimers via plant peroxidases and/or photodimerizationinitiated by sunlight. Further, phenolic acids have been shown tofunction as cross-links between cell wall polysaccharides and thephenylpropanoid lignin polymer. The covalent attachment of lignin towall polysaccharides and the crosslinking of xylan chains withinhemicellulose limit overall polysaccharide bioavailability resulting insignificant amounts of undigested fiber in animal feedstuffs, poorbioconversion of agricultural residue into useful products andincomplete processing of grains.

Enzyme hydrolysis of xylan to its monomers requires the participation ofseveral enzymes with different functions. These are classified in twogroups based on the nature of the linkages that they cleave. The firstgroup of enzymes is hydrolases (EC 3.2.1) involved in the hydrolysis ofthe glycosidic bonds of xylan. These include endo-xylanases (EC 3.2.1.8)which randomly dismember the xylan backbone into shorterxylooligosaccharides; β-xylosidase (EC 3.2.1.37) which cleave thexylooligosaccharides in an exo-manner producing xylose;α-L-arabinofuranosidase (EC 3.2.1.55); and α-glucoronidase (EC 3.2.1.1)which remove the arabinose and 4-O-methylglucuronic acid substituents,respectively, from the xylan backbone. The second group includes enzymesthat hydrolyze the ester linkages (esterase, EC 3.1.1) between xyloseunits of the xylan polymer and acetyl groups (acetyl xylan esterase, EC3.1.1.6) or between arabinosyl groups and phenolic moieties such asferulic acid (feruloyl esterase) and p-coumaric acid (coumaroylesterase).

Faulds et al., reported two forms of ferulic acid esterase isolated fromAspergillus niger. The different esterases were distinguished on thebasis of molecular weight and substrate specificity (Faulds et al.,Biotech. Appl. Biochem., vol. 17, pp. 349-359 (1993)). Brezillon et al.disclosed the existence of at least two cinnamoyl esterases which werebelieved to be distinct from the ferulic acid esterases shown in theprior art (Brezillon et al., Appl. Microb. Biotechnol., vol. 45, pp.371-376 (1996)). A ferulic acid esterase called FAE-III was isolatedfrom Aspergillus niger CBS 120.49 and shown to act together withxylanase to eliminate nearly all of the ferulic acid and low molecularmass xylooligosaccharides in a wheat bran preparation; ferulic acid wasalso removed without the addition of xylanase, albeit at a lower level.Faulds et al. further isolated and partially characterized FAE-III fromAspergillus niger CBS 120.49 grown on oat spelt xylan (Faulds et al.,Microbiology, vol. 140, pp. 779-787 (1994)) and showed it to have a plof 3.3, a molecular weight of 36 kD (SDS-PAGE) and 14.5 kD (GelFiltration method), a pH optimum of 5 and a temperature optimum of55-60° C.; microcrystalline cellulose binding was also detected. Theauthors theorized that FAE-II may be a proteolytically modified FAE-III.Recently, the various known ferulic acid esterases derived fromAspergillus niger have been distinguished based on their distinctsubstrate specificity and it was noted that FAE-II and FAE-III wereunable to release ferulic acid from sugar beet pulp (Brezillon et al.,supra).

Nonetheless, despite the characterization work which has been directedto Aspergillus niger esterases, the art remains in need of additionalesterases for its various applications. Further, those of skill in theart have thus far failed to discover a nucleotide sequence which can beused to produce more efficient genetically engineered organisms capableof expressing such esterases in large quantities suitable for industrialproduction. However, a pressing need exists for the development of anesterase expression system via genetic engineering which will enable thepurification and utilization of working quantities of relatively pureenzyme.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide for novel esteraseproteins and DNA encoding such proteins.

It is an object of the present invention to provide for a method ofisolating DNA from many different species, which DNA encodes proteinhaving esterase activity.

It is a further object of the present invention to provide for anesterase which is produced by a suitable host cell which has beentransformed by the DNA encoding the esterolytic activity.

The present invention provides for a purified 38 kD esterase which isderived from Aspergillus niger. Further, a DNA sequence coding for the38 kD esterase comprising a DNA as shown in FIG. 4 (SEQ. ID NO: 27); aDNA which encodes the amino acid sequence also shown in FIG. 4 (SEQ. IDNO: 28); a DNA which encodes an esterase which comprises an amino acidsegment which differs from the sequence in FIG. 4, provided that the DNAencodes a derivative of the 38 kD esterase specifically describedherein; and a DNA which encodes an esterase that comprises an amino acidsegment which differs from the sequence in FIG. 4, provided that the DNAhybridizes under low-stringency conditions and/or standard stringencyconditions, as defined below, with a DNA comprising all or part of theDNA in FIG. 4 are provided. The present invention further encompassesvectors which include the DNA sequences described above, host cellswhich have been transformed with such DNA or vectors, fermentationbroths comprising such host cells and esterase proteins encoded by suchDNA which are expressed by the host cells. Preferably, the DNA of theinvention is in substantially purified form and is used to prepare atransformed host cell capable of producing the encoded protein productthereof. Additionally, polypeptides which are the expression product ofthe DNA sequences described above are within the scope of the presentinvention.

The enzyme of the instant invention has application as a supplement toan animal feed; in a process for treating fabric; to improve themechanical properties of dough and the end product of baking of foods;in the modification of polysaccharides to give novel properties, e.g.,gums; and in the processing grains. Further, the enzyme also hasapplication in processing of plant materials for the release of freephenolic groups for use as an antioxidant, photoprotector,anti-inflammatory and/or anti-microbial agent which find use in personalcare products such as cosmetics and as an aid in the conversion ofchemical feed stocks to valuable specialty chemicals, food additives andflavorings.

An advantage of the present invention is that a DNA has been isolatedwhich provides the capability of isolating further DNAs which encodeproteins having esterolytic activity.

Another advantage of the present invention is that, by virtue ofproviding a DNA encoding a protein having esterolytic activity, it ispossible to produce through recombinant means a host cell which iscapable of producing the protein having esterolytic activity inrelatively large quantities.

Yet another advantage of the present invention is that commercialapplication of proteins having esterolytic activity is made practical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a western blot following SDS-PAGE gel showing thefragmentation of FAE under denaturing conditions.

FIG. 2 illustrates the DNA sequence (SEQ. ID NO: 25) with deducedintrons and amino acid sequence (SEQ. ID NO: 26) of a 650 base pairfragment corresponding to the gene encoding a 38 kD esterase isolatedfrom Aspergillus niger.

FIG. 3 illustrates a restriction map of a DNA fragment containing thegene encoding the 38 kd esterase.

FIGS. 4A-4E illustrate the complete DNA (SEQ. ID NO: 27), withhighlighting to point out the signal sequence, intron and variousrestriction endonuclease sites, and amino acid sequence (SEQ. ID. NO:28) corresponding to the gene encoding the 38 kD esterase isolated fromAspergillus niger.

FIG. 5 illustrates the DNA sequence of the gene encoding the 38 kDesterase (SEQ. ID. NO: 29).

FIG. 6 illustrates a southern blot gel showing hybridization between aDNA probe derived from the 38 kD esterase of the invention and severalother filamentous fungi (“gel 1”).

FIG. 7 illustrates a southern blot gel showing hybridization between aDNA probe derived from the 38 kD esterase of the invention and severalother filamentous fungi (“gel 2”).

DETAILED DESCRIPTION OF THE INVENTION

“Esterase” or “esterolytic activity” means a protein or peptide whichexhibits esterolytic activity, for example, those enzymes havingcatalytic activity as defined in enzyme classification EC 3.1.1.Esterolytic activity may be shown by the ability of an enzyme or peptideto cleave ester linkages, for example, feruloyl, coumaroyl or acetylxylan groups, from organic compounds in which they are known to exist,e.g., primary and secondary cell walls. Preferably, the esterasecomprises an esterolytic activity which cleaves the ester linkage ofphenolic esters such as:[5O-((E)-feruloyl)-α-L-arabinofuranosyl](1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose(also known as FAXX);[5-O-((E)-feruloyl)-α-L-arabinofuranosyl](1→3)-O-β-D-xylopyranose (alsoknown as FAX);O-β-D-xylopyranosyl-(1→4)-O-[5-O-((E)-feruloyl)-α-arabinofuranosyl-(1→3)]-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose(also known as FAXXX);[5-O-((E)-p-coumaroyl)-α-L-arabinofuranosyl](1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose(also known as PAXX);[5-O-((E)-p-coumaroyl)-α-L-arabinofuranosyl](1→3)-O-β-D-xylopyranose(also known as PAX);O-β-D-xylopyranosyl-(1→4)-O-[5-O-((E)-p-coumaroyl)-α-arabinofuranosyl-(1→3)]-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose(also known as PAXXX) and other ester linked phenolic oligosaccharidesas are known in the art. Such esterases are generally referred to asferulic acid esterase (FAE) or enzymes having feruloyl esteraseactivity. It has surprisingly been discovered that an esterase havingferulic acid esterase activity which may be purified from Aspergillusniger, as described herein, and having an amino acid sequence as shownin FIG. 4, further has activity on sugar beet pulp and also proteolyticand lipolytic activity. Thus, according to a particularly preferredembodiment of the present invention, an esterase and/or a DNA encodingthat esterase is provided which esterase also has lipolytic and/orproteolytic activity. Accordingly, the esterase of the invention havingmeasurably significant esterolytic activity on feruloyl and coumaroylesters also has proteolytic and lipolytic activity.

Preferably, the esterase and/or DNA encoding the esterase according tothe present invention is derived from a fungus, more preferably from ananaerobic fungus and most preferably from Aspergillus spp., e.g.,Aspergillus niger. Thus, it is contemplated that the esterase or the DNAencoding the esterase according to the invention may be derived fromAbsidia spp.; Acremonium spp.; Actinomycetes spp.; Agaricus spp.;Anaeromyces spp.; Aspergillus spp., including A. auculeatus, A. awamori,A. flavus, A. foetidus, A. fumancus, A. fumigatus, A. nidulans, A.niger, A. oryzae, A. terreus and A. versicolor; Aeurobasidium spp.;Cephalosporum spp.; Chaetomium spp.; Coprinus spp.; Dactyllum spp.;Fusarium spp., including F. conglomerans, F. decemcellulare, F.javanicum, F. lini, F.oxysporum and F. solani; Gliocladium spp.;Humicola spp., including H. insolens and H. lanuginosa; Mucor spp.;Neurospora spp., including N. crassa and N. sitophila; Neocallimastixspp.; Orpinomyces spp.; Penicillium spp; Phanerochaete spp.; Phlebiaspp.; Piromyces spp.; Pseudomonas spp.; Rhizopus spp.; Schizophyllumspp.; Streptomyces spp; Trametes spp.; and Trichoderma spp., includingT. reesei, T. longibrachiatum and T. viride; and Zygorhynchus spp.Similarly, it is envisioned that an esterase and/or DNA encoding anesterase as described herein may be found in bacteria such asStreptomyces spp., including S. olivochromogenes; specifically fiberdegrading ruminal bacteria such as Fibrobacter succinogenes; and inyeast including Candida torresii, C. parapsilosis; C. sake; C.zeylanoides; Pichia minuta; Rhodotorula glutinis; R. mucilaginosa; andSporobolomyces holsaticus.

According to a preferred embodiment of the invention, the esterase is ina purified form, i.e., present in a particular composition in a higheror lower concentration than exists in a naturally occurring or wild typeorganism or in combination with components not normally present uponexpression from a naturally occurring or wild type organism.

“Expression vector” means a DNA construct comprising a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of the DNA in a suitable host. Such controlsequences may include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable ribosome-binding sites on the mRNA, and sequences which controltermination of transcription and translation. Different cell types arepreferably used with different expression vectors. A preferred promoterfor vectors used in Bacillus subtilis is the AprE promoter; a preferredpromoter used in E. coli is the Lac promoter and a preferred promoterused in Aspergillus niger is glaA. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may, under suitable conditions, integrate into thegenome itself. In the present specification, plasmid and vector aresometimes used interchangeably. However, the invention is intended toinclude other forms of expression vectors which serve equivalentfunctions and which are, or become, known in the art. Thus, a widevariety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences such as various knownderivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.coli including col E1, pCR1, pBR322, pMb9, pUC 19 and their derivatives,wider host range plasmids, e.g., RP4,phage DNAs e.g., the numerousderivatives of phage λ, e.g., NM989, and other DNA phages, e.g., M13 andfilamentous single stranded DNA phages, yeast plasmids such as the 2 μplasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in animal cells and vectors derived from combinationsof plasmids and phage DNAs, such as plasmids which have been modified toemploy phage DNA or other expression control sequences. Expressiontechniques using the expression vectors of the present invention areknown in the art and are described generally in, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press (1989). Often, such expression vectors including theDNA sequences of the invention are transformed into a unicellular hostby direct insertion into the genome of a particular species through anintegration event (see e.g., Bennett & Lasure, More Gene Manipulationsin Fungi, Academic Press, San Diego, pp. 70-76 (1991) and articles citedtherein describing targeted genomic insertion in fungal hosts,incorporated herein by reference).

“Host strain” or “host cell” means a suitable host for an expressionvector comprising DNA according to the present invention. Host cellsuseful in the present invention are generally procaryotic or eucaryotichosts, including any transformable microorganism in which expression canbe achieved. Specifically, host strains may be Bacillus subtilis,Escherichia coli, Trichoderma longibrachiatum, Saccharomyces cerevisiaeor Aspergillus niger, and preferably Aspergillus niger. Host cells aretransformed or transfected with vectors constructed using recombinantDNA techniques. Such transformed host cells are capable of bothreplicating vectors encoding esterase and its variants (mutants) orexpressing the desired peptide product.

“Derivative” means a protein which is derived from a precursor protein(e.g., the native protein) by addition of one or more amino acids toeither or both the C- and N-terminal end, substitution of one or moreamino acids at one or a number of different sites in the amino acidsequence, deletion of one or more amino acids at either or both ends ofthe protein or at one or more sites in the amino acid sequence, orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of an enzyme derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative enzyme.The derivative of the invention includes peptides comprising alteredamino acid sequences in comparison with a precursor enzyme amino acidsequence (e.g., a wild type or native state enzyme), which peptidesretain a characteristic enzyme nature of the precursor enzyme but whichhave altered properties in some specific aspect. A “derivative” withinthe scope of this definition will retain generally the characteristicesterolytic activity observed in the native or parent form to the extentthat the derivative is useful for similar purposes as the native orparent form, However, it is further contemplated that such derivativesmay have altered substrate specificity, e.g., greater or lesser affinityfor a specific substrate such as feruloyl, cinnamoyl or coumaroylgroups, or modified pH, temperature or oxidative stability. Thederivative of the invention may further be produced through chemicalmodification of the precursor enzyme to alter the properties thereof.

Hybridization is used herein to analyze whether a given fragment or genecorresponds to the esterase described herein and thus falls within thescope of the present invention. The hybridization assay is essentiallyas follows: Genomic DNA from a particular target source is fragmented bydigestion with a restriction enzyme(s), e.g., EcoR I, Hind III, PinA I,Mlu I, Spe I, Bgl II, Ppu10 I, Mfe I, Nco I, Bln I, Eag I and Xma I(supplied by New England Biolabs, Inc., Beverly, Mass. and BoehringerMannheim) according to the manufacturer's instructions. The samples arethen electrophoresed through an agarose gel (such as, for example, 0.7%agarose) so that separation of DNA fragments can be visualized by size.The gel may be briefly rinsed in distilled H₂O and subsequentlydepurinated in an appropriate solution for 30 minutes (such as, forexample, 0.25M HCl) with gentle shaking followed by denaturation for 30minutes (in, for example, 0.4 M NaOH) with gentle shaking. The DNAshould then be transferred onto an appropriate positively chargedmembrane, for example the Maximum Strength Nytran Plus membrane(Schleicher & Schuell, Keene, N.H.), using a transfer solution (such as,for example, 0.4 M NaOH). After the transfer is complete, generally atabout 2 hours or greater, the membrane is rinsed and air dried at roomtemperature after using a rinse solution (such as, for example,2×SSC[2×SSC=300 mM NaCl, 30 mM trisodium citrate]). The membrane shouldthen be prehybridized (for approximately 2 hours or more) in a suitableprehybridization solution (such as, for example, an aqueous solutioncontaining per 100 mls: 20-50 mls formamide, 25 mls of 20×SSPE(1×SSPE=0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7), 2.5 mls of 20%SDS, 1 ml of 10 mg/ml sheared herring sperm DNA and 21.5 ml distilledH₂O). As would be known to one of skill in the art, the amount offormamide in the prehybridization solution may be varied depending onthe nature of the reaction obtained according to routine methods. Thus,a lower amount of formamide may result in a more complete gel in termsof identifying hybridizing molecules than the same procedure using alarger amount of formamide. On the other hand, a strong hybridizationband may be more easily visually identified by using more formamide.

The DNA probe derived from the sequence in FIGS. 4 or 5 should beisolated by electrophoresis in 1% agarose, the fragment excised from thegel and recovered from the excised agarose. This purified fragment ofDNA is then random prime ³²P labeled (using, for example, the Megaprimelabeling system according to the instructions of the manufacturer(Amersham International plc, Buckinghamshire, England)). The labeledprobe is denatured by heating to 95° C. for 5 minutes and immediatelyadded to the prehybridization solution above containing the membrane.The hybridization reaction should proceed for an appropriate time andunder appropriate conditions, for example, for 18 hours at 37° C. withgentle shaking. The membrane is rinsed (for example, in 2×SSC/0.3% SDS)and then washed with an appropriate wash solution and with gentleagitation. The stringency desired will be a reflection of the conditionsunder which the membrane (filter) is washed.

Specifically, the stringency of a given reaction (i.e., the degree ofhomology necessary for successful hybridization) will depend on thewashing conditions to which the filter from the Southern Blot issubjected after hybridization. “Low-stringency” conditions as definedherein will comprise washing a filter from a Southern Blot with asolution of 0.2×SSC/0.1% SDS at 20° C. for 15 minutes.“Standard-stringency” conditions comprise a further washing stepcomprising washing the filter from the Southern Blot a second time witha solution of 0.2×SSC/0.1% SDS at 37° C. for 30 minutes.

FIGS. 4 and 5 illustrates the amino acid sequence and DNA sequence of anovel esterase derived from Aspergillus niger . The isolated esterasehas a molecular weight of about 38 kD (as shown on SDS-PAGE), a pl ofabout 2.8 (as shown on IEF), a pH optimum of about 5.1 on methylferulate, a temperature optimum of about 55° C. and activity oncoumaroyl and feruloyl esters, and sugar beet pulp. The FAE gene shownin FIG. 4 (SEQ. ID NO: 27) is approximately 2436 base pairs in lengthincluding deduced intron sequence and, if expressed, will encode theherein identified esterase from Aspergillus niger (hereinafter the “38kD esterase”). For the purposes of the present invention, the term “38kD esterase” means an esterase derived from Aspergillus nigercorresponding to the esterase specifically exemplified herein. The DNAprovided in FIGS. 4 or 5 will be useful for obtaining homologousfragments of DNA from other species, and particularly from anaerobicfungi, which encodes an enzyme having esterolytic activity.

The DNA sequences of the present invention may be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employed in that expression vector totransform an appropriate microbial host according to techniques wellestablished in the art. The polypeptides produced on expression of theDNA sequences of this invention may be isolated from the fermentation ofanimal cell cultures and purified in a variety of ways according to wellestablished techniques in the art. One of skill in the art is capable ofselecting the most appropriate isolation and purification techniques.

The esterase isolated according to the present invention is useful inapplications in which it is desired to remove phenolic constituents ofxylan oligosaccharides. For example, esterases may be applied to improveanimal, and possibly human, nutrition as the digestibility of foragecell walls appears to be dependent on the phenolic content of theforage. Furthermore, esterases could be applied in the pulp and paperindustry as hydrolysis of phenolic ester linked moieties from lignin maycontribute to solubilization of s the lignin and also may contribute tohydrolysing lignin/hemicellulose linkages. Esterases may be of potentialuse in the synthesis of carbohydrate derivatives and in thebioconversion of agricultural residue to fermentable sugars and freephenolic acid useful as an antioxidant, photoprotectant and/orantimicrobial in foods and personal care products; as a feed stock forconversion to flavors (such as vanillin) biopolymers, and valuablechemicals. Esterases have also been implicated in the finishing oftextile fibers (see e.g., PCT Publication No. 96/16136). The activity ofesterases toward multiple substrates present in many dirt based stains,their activation by surfactants and specificity toward phenolicssuggests that esterases may also be of value in detergents. Theavailability of relatively large quantities of esterase facilitated bythe present invention will enable the development of additional valuableapplications.

The invention will be explained further below in the accompanyingexamples which are provided for illustrative purposes and should not beconsidered as limitative of the invention.

EXAMPLES Example 1 Purification and Isolation of Peptides ComprisingFerulic Acid Esterase Activity and Design of Degenerate DNA Fragmentsfor PCR

A fermentation broth from Aspergillus niger was filtered (0.8 μm) and 10ml transferred into a centrifuge tube (50 ml) at room temperature.Saturated (NH₄)₂SO₄ was added to give a final concentration of 60%. Thesolution was mixed and stored for approximately one hour at 4° C. thencentrifuged at 1500×g for 20 minutes at 4° C. The supematant was removedand the pellet resuspended in distilled water. Four tubes prepared asabove were combined and diluted to approximately 200 ml with ammoniumsulfate (2 M) to give a final concentration of 1.2 M ammonium sulfate,the pH was adjusted to pH 7.4 by the addition of Tris-HCl (200 mM).

The enzyme sample was chromatographed by hydrophobic interactionchromatography (Poros® HPEM phenyl ether, Perseptive BioSystems,perfusion chromatography, 12×30 cm). The column was connected to aBioCad® Perfusion Chromatography Workstation (Perseptive BioSystems) andequilibrated with 5 column volumes Tris-HCl (50 mM, pH 7.4) plus 1.2 Mammonium sulfate. The sample (205 ml) was applied to the column andseparated at a flow rate of 30 ml/min with a linear gradient from 1.2 Mto 200 mM ammonium sulfate over 20 column volumes. Fractions (15 ml)were collected during the gradient phase of the separation and assayedfor FAE activity with methyl ferulate by the method of Faulds andWilliamson (1994,Microbiology 140: 779-787). Four fractions eluting at750 mM ammonium sulfate contained 83% of the starting FAE activity.Active fractions were pooled (60 ml), dialysed by ultrafiltration intostart buffer for next chromatographic step (10 kDa membrane, 20 L of 25mM sodium acetate buffer pH 5.0). The sample was concentrated to 10 mlin preparation for ion exchange chromatography.

Ion exchange chromatography was performed using a MonoQ strong anionexchanger (MonoQ®, HR 10/10, Pharmacia Biotechnology) connected toBioCAD Perfusion Chromatography Workstation and equilibrated with 25mMsodium acetate (pH 5.0). The sample (10 ml) was applied to the columnand eluted at a flow rate of 5 ml/min with a linear NaCl gradient (0-500mM) over 15 column volumes. Fractions (5 ml) were collected during thegradient and assayed for FAE activity (activity against feruloylesters). FAE activity eluted as a single peak at 155 mM NaCl and wascollected in one fraction. The sample was concentrated (Centricon, 10kDa) to 1.5 ml.

High performance size exclusion chromatography (HPSEC) was carried outusing two Superdex 75 columns (10/30 HR, Pharmacia Biotechnology)connected in tandem on a BioCAD Perfusion Chromatography Workstation.The columns were equilibrated with 10 column volumes sodium acetatebuffer (25 mM, pH 5.0) containing 125 mM NaCl and 0.01% triton X-100.The columns were calibrated for determination using protein standards ofknown molecular mass (Bio-RAD gel filtration standards, and Sigma gelfiltration. standards). Samples (500 μl) were applied and separated at aflow rate of 750 μl/min. Fraction (1 ml) were collected. The FAEactivity eluted as a single peak corresponding to a molecular mass ofabout 32 kDa.

Upon native PAGE of a desalted active fraction from HPSEC a singleprotein band was observed. The isolelectric point of the FAE wasdetermined using Phast gel Dry IEF equilibrated with a solutionampholyte, (20% final concentration, containing a mix of pl 2-4, 80%, pl3-10 20%) Glycerol (10%) for 1 hour. Separation was performed as permanufacturers recommendation (Pharmacia Biotechnology, Dry IEFinstruction bulletin) and stained with coomassie R-250. The samplemigrated as a single protein band with an isolelectric point of about2.8.

Western Blots of the HPSEC FAE sample were performed using PVDFmembranes (0.2 μm pore size) a Novex mini-gel apparatus for obtainingN-terminal amino acid sequence by methods recommended by manufacturer. Asample of the FAE in dialysed into buffer (5 mM MES pH 5.8).

The resultant purified protein was placed in an aqueous solution forpeptide sequence analysis according to standard methods. Briefly thepeptides were digested in solution with the following sequencing gradeproteases:

Lys-C—200 μl reaction buffer comprising 100 mM ammonium bicarbonate and2-4 μg of enzyme, pH 8.0, overnight at 37° C.;

Arg-C—200 μl reaction buffer comprising 20 mM Tris and 4 μg of enzyme,pH 7.5+1.5 mM

CaCl₂+2 mM DTT overnight at 37° C.;

Glu-C—digest buffer for on-blot digests was 50 mM ammonium bicarbonatewith 4 μg enzyme, 10% acetonitrile and 1% reduced triton X-100;

CNBr cleavage—was conducted by dissolving enzyme sample in 200 μl of 70%formic acid in water and CNBr crystals added in sufficient quantity toproduce methionine cleavage.

The digested peptides were then concentrated to approximately 100 μl andloaded is directly onto a reverse-phase HPLC (Phenomenex Primesphere C18column, 250×2.0 mm). Reverse phase separations were carried out usingApplied Biosystems 140A solvent delivery system. Buffers used were 0.1%TFA in water (A), 70% acetonitrile in water+0.070% TFA (B); flow ratewas 150 μl per minute with a gradient as follows: 0 minutes—5% Buffer B,10 minutes—10% Buffer B, 80 minutes—80% Buffer B, 85 minutes—100% BufferB, 90 minutes—100% Buffer B.

CNBr digests are treated as follows: water is added to the solution andthe whole volume concentrated to 100 μl in a speed-vac. Further water isadded to approximately 1 ml and this dried again to about 100 μl. Thisremoves the majority of the formic/CNBr.

Various peptide fragments obtained as described above were analyzed todetermine their sequence and for subsequent development of degenerateprobes for use in cloning the gene encoding the 38 kD esterase from thegenome of the donor organism. Peptide sequence analysis of the 38 kDesterase was problematic due to cycles containing mixed signalsindicating the presence of multiple polypeptides in the analyzed sample.Protein sequencing resulted in an N-terminal sequence and severaladditional peptide fragments as follows:

ASTQGISEDLYSRLVEMATISQAAYXDLLNIP (SEQ. ID NO:1) XTVGFGPY (SEQ. ID NO:2)FGLHLXQXM (SEQ. ID NO:3) XISEDLYS (SEQ. ID NO:4) YIGWSFYNA (SEQ. IDNO:5) GISEDLYXXQ (SEQ. ID NO:6) XISESLYXXR (SEQ. ID NO:7) GISEDLY (SEQ.ID NO:8) LEPPYTG (SEQ. ID NO:9) XANDGIPNLPPVEQ (SEQ. ID NO:10) YPDYALYK(SEQ. ID NO:11)

From these fragments, suitable degenerate probes for hybridization anduse as PCR primers were produced and fragments were obtained which werebelieved to be derived from the gene encoding the 38 kD esterase.However, sequencing of the fragments obtained in this manner (550 and100 base pairs) showed that the fragments were merely artifacts of PCRand were not of use in cloning the 38 kD esterase. Additional analysisof 2 different probes derived from 2 protein sequences isolated as aboveresulted in similar lack of success. From these results, it wasdetermined that routine protein purification and peptide sequencingprocedures were insufficient to obtain suitable peptide fragments forthe preparation of degenerate DNA probes.

The inventors herein hypothesized that a specific property of theprotein or the purified protein composition was preventing obtainingpurified representative protein. To test this theory, the productprotein from above was analyzed via isolelectric focusing gel at pH 2-4under various conditions. Protein samples taken from purification stepsalong the purification method described above appeared to be a singleband of highly purified protein. A second analysis was performed inwhich the purified protein was subjected to denaturing conditions of anSDS-PAGE and the results western blotted. As shown in FIG. 1, theresultant protein showed a number of bands indicating either somedegeneration of the protein or other compounds hidden during the IEFgel. Sequencing of each of the numerous bands showed that each possessedan identical N-terminal sequence and that proteolysis appeared to beoccurring from the carboxy terminal.

From the data, the inventors herein hypothesized that numerous fragmentsmay be appearing due to carboxy terminal proteolytic clipping within themolecule itself upon unfolding of the protein in reduced SDS buffer.Unfolding of the 38 kD esterase may expose the previously internalhydrophobic residues, e.g., tyrosine, tryptophan and phenylalanine,providing a structurally similar substrate to the ester linked feruloylgroup which would be recognized in the active site of the 38 kD esteraseallowing for hydrolysis peptide. This result was highly unpredictabledue to the fact that the heretofore observed enzymatic action of theisolated protein was esterolytic and not proteolytic. In any event, theinventors herein theorized that if protein denaturing conditions (i.e.,unfolding of the peptide chain) were avoided, internal clipping may beavoided. To effect this, the purified protein from the anion exchangechromatography step was further chromatographed using high resolutionsize exclusion chromatography (HPSEC as detailed above).

The HPSEC purified 38 kD esterase was separated by SDS-PAGE and aWestern blot onto a PVDF membrane was performed for sequencing“on-blot”. Digests directly from the blots were prepared as follows:neat TFA is added to the blot containing solution to give a final volumecontaining 50% TFA. This solution is then sonicated for 5 minutes. Theliquid (but not the blot pieces) is removed and a solution of 50%acetonitrile in 0.1% TFA is added. The sample is sonicated again for 5minutes. The liquid was removed and replaced by a final wash of 0.1% TFAin water and a final 5 minutes sonication. All wash solutions werepooled and concentrated down to approximately 100 μl. This methodallowed for the polypeptides resulting from enzymatic digests to becollected without further proteolysis by 38 kD esterase immobilized onthe membrane. In this way, single polypeptides suitable for sequenceanalysis were obtained due to 38 kD esterase being immobilized on thePVDF thus preventing carboxy terminal proteolytic clipping and thepresence of mixed amino acid signals during each cycle of sequencing.

When this procedure was followed, a number of fragments which wereappropriate for the design of degenerate DNA fragments were produced.

Example 2 Isolation of A 650 Base Pair Fragment Corresponding to FAEGene

Based on the peptide fragments obtained in Example 1 after the proteinclipping problem had been solved, the gene encoding the 38 kD esterasewas cloned by amplifying the gene from its genome using polymerase chainreaction and appropriately designed degenerate oligonucleotide primers.Primers were designed based upon partial amino acid sequences offragmented 38 kD esterase protein. Amplification of three fragments fromthe 38 kD esterase gene was obtained using the following fouroligonucleotide primers (the oligonucleotide primers were designed basedupon the underlined peptide sequence following the oligonucleotideprimers. The following abbreviations were used to identify wobbleposition alternates: I=inosine, W=A/T, S=C/G, R=A/G, Y=T/C, H=A/T/C,D=A/G/T, X=A/T/G/C.

Sense primer 11: CGGGAATTCGCIWSIACICARGGXAT (SEQ ID. NO:12)

Derived from: ASTQGISEDLYSRLVEMATISQAAYADLLNIP (SEQ ID. NO:13)

Sense primer 7: CGGGAATTCTAYTAYATHGGITGGGT (SEQ. ID NO:14)

Derived from: VHGGYYIGWVSVQDQV (SEQ. ID NO:15)

Anti-sense primer 8: CGGGAATTCACCCAICCDATRTARTA (SEQ. ID NO:16)

Derived from: VHGGYYIGWVSVQDQV (SEQ. ID NO:17)

Anti-sense primer 2: CGGGAATTCTTIGGIATICCRTCRTT (SEQ. ID NO:18)

Derived from: TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ. ID NO:19)

Two primers enabled deduction of putative amplified DNA fragmentsencoding the 38 kD esterase:

Anti-sense primer 3: CGGGAATTCATICCRTCRTTIGCRTG (SEQ. ID NO:20)

Derived from: TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ. ID NO:21)

Anti-sense primer 12: CGGGAATTCGCYTGRAAIGCRTCIGTCAT (SEQ. ID NO:22)

Derived from: (M)TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ. ID NO:23)

An EcoR I restriction endonuclease recognition site and a “GC” clamp wasincluded at the 5′ end of all primers to facilitate cloning of amplifiedfragments into the plasmid vector pUC18. PCR reaction included placingthe following into “Hot Start” (Molecular Bio-Products, Inc., San Diego,Calif.) tubes in the order provided:

1 μl 500 ng/μl sense primer

1 μl 500 ng/μl anti-antisense primer

2 μl nucleotide mix (10 mM each dNTP)

5 μl 10× PCR Buffer

41 μl distilled water

Heated at 95° C. for 90 seconds, placed onto ice for 5 minutes.

5 μl 10× PCR Buffer

43 μl distilled water

1 μl Aspergillus niger genomic DNA

1 μl Taq DNA Polymerase (Boehringer Mannheim, 5 U/μl)

Amplification was carried out in a Minicycler Model PTC -150 (MJResearch Inc., Watertown, Mass.). Amplification conditions followed asequential pattern of: 95° C. for five minutes; 40° C. for 90 seconds;72° C. for 3 minutes; and 28 cycles of 94° C. for 1 minute, 40° C. for90 seconds, and 72° C. for 3 minutes for 28 cycles. A final extensionstep of 72° C. at 2 minutes was included.

The primers were used for PCR amplification in the following pairedcombinations: 11-2, 11-3, 11-8, 11-12, 7-2, 7-3 and 7-12. Each primercombination produce multiple DNA bands upon agarose electrophoresis.Major DNA bands for the PCR products of primers pairs 7-2, 7-3 and 7-12were present at around 350, 350 and 300 base pairs respectively asvisualized on a 3% NuSieve (FMC Corp.) agarose electrophoresis gel.Antisense primers 2, 3 and 12 were designed to the same continuouspeptide fragment: (M)TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ. ID NO: 24).Anti-sense primers 2 and 3 code for nearly the same stretch of DNA,their 3′ ends being offset by only 6 bases. As antisense primer 12corresponds to amino acids that are upstream of primers 2 and 3, the 3′end of antisense primer 12 is offset by approximately 60 base pairs fromprimers 2 and 3. Therefore, the lengths of the PCR bands wereapproximately consistent with a continuous stretch of DNA encoding the38 kD esterase. Additionally the primer pairs 11-2, 11-3 and 11-12produced bands of approximately 650, 650 and 600 base pairsrespectively. These lengths were approximately consistent withamplification of a piece of DNA encoding the 38 kD esterase.

The PCR amplification products were digested with EcoRI, ligated intothe cloning vector pUC18 and then transformed into E. coli. The clonedPCR products were sequenced. Sequencing of the product of primers 11-2revealed a 650 base pair. DNA sequence shown in FIG. 2 (SEQ. ID NO:25)which upon translation codes for 197 amino acids. A total of 155residues corresponded to nine sequenced peptide fragments of 38 kDesterase protein. A putative 57 base pair intron exists containingsplicing sequences of GTATGC at the 5′ site, an internal lariat sequenceof CACTAACT, and TAG at the 3′ splice site. Furthermore a product ofprimers 11-8 when sequenced reveals approximately the first 314 bases(5′-3′) of the 650 base pair 11-2 fragment. A 350 base pair product ofprimers 7-2 revealed DNA that corresponds in sequence to the second halfof the 650 base pair 11-2 fragment.

Example 3 Obtaining Genomic DNA from Aspergillus Niger for Cloning

A preserved culture of Aspergillus niger was grown on Potato DextroseAgar (PDA) at 30° C. Approximately 2 cm² of the fungi grown on PDA wasinoculated into 50 ml of Yeast Extract Glucose media in a 250 ml baffledflask and incubated at 33° C. in a rotary shaker at a speed of 300 RPMfor 24 hours. The mycelia was harvested through miracloth, squeezed dry,immediately frozen in liquid nitrogen and ground with ½ teaspoon of sandin a mortar and pestle for approximately two minutes. The genomic DNAwas extracted from the ground mycelia using a modification ofInvitrogen's Easy-DNA Genomic Isolation Kit. The ground, frozen myceliawas immediately transferred to a centrifuge tube to which 3.5 mls ofSolution A was added, followed by vortexing and a 10 minute incubationat 65° C. Next, 1.5 mls of Solution B was added, followed by vortexing.5 mls of chloroform was added, followed by vortexing until the viscositydecreased and the mixture was homogeneous. The mixture was centrifugedat 15,000×G at 4° C. for 20 minutes. The upper phase was transferredinto a new tube and then precipitated with two volumes of 95% ethanol.The precipitation reaction was incubated on ice for 30 minutes. Theprecipitation DNA was pelleted by centrifugation at 15,000×G at 4° C.for 15 minutes. The ethanol was removed. The DNA pellet was washed with25 mls of 70% ethanol and the mixture was centrifuged at 15,000×G at 40°C. for 5 minutes. The 70% ethanol was removed and the pellet allowed toair dry for 5 minutes The extracted DNA was suspended in a volume of 500μl of TE, RNase was added to a final concentration of 4 μg/ml. Thisextracted genomic DNA was used in PCR amplification of the DNA fragmentsencoding 38 kD esterase.

Example 4 Using the Obtained 650 Base Pair Fragment to Isolate DNAEncoding Homologous Enzymes from Aspergillus or Other Species

One particularly effective method of obtaining clones of homologousgenomic DNA is by construction and screening of a subgenomic libraries.Briefly, and as described in more detail below, this method involvescutting the genomic DNA to completion with appropriate restrictionendonucleases, performing Southern hybridization with the 650 base pairfragment as a probe, ligating the appropriately sized fragments into aplasmid vector, transforming the plasmid into E. coli and then southernprobing the colonies with the 650 base pair fragment to obtain a genomicclone. These techniques are known in the art and are described inCurrent Protocols in Molecular Biology, supra.

To obtain clones of a vector comprising the gene encoding the entire 38kD esterase protein, Aspergillus niger genomic DNA was prepared as inExample 3. The genomic DNA was fragmented by digestion with a number ofrestriction enzymes: EcoR I, Hind III, PinA I, Mlu I, Spe I, Bgl II,Ppu10 I, Mfe I, Nco I, Bin I, Eag I and Xma I (supplied by New EnglandBiolabs, Inc., Beverly, Mass. and Boehringer Mannheim). The reactionconditions combined 3 μl of genomic DNA, 2 μl of the appropriate 10×restriction endonuclease buffer (according to the manufacturersinstructions), 2 μl of restriction enzyme (at 10 units/μl), 13 μl ofdistilled water; the reaction proceeded at 37° C. for 6 hours. Thesamples were then electrophoresed through a 0.7% agarose gel so thatseparation of DNA fragments could be visualized between a size of 1 kbto >12 kb. The gel was briefly rinsed in distilled H₂O and subsequentlydepurinated for 30 minutes in a solution of 0.25M HCl with gentleshaking followed by denaturation for 30 minutes in a solution of 0.4 MNaOH with gentle shaking. The DNA was then transferred onto a positivelycharged Maximum Strength Nytran Plus membrane (Schleicher & Schuell,Keene, N.H.) using a solution of 0.4 M NaOH as transfer solution. Afterthe transfer was complete, >2 hours, the membrane was rinsed in 2×SSCand air dried. The membrane was then prehybridized for 8 hours in aprehybridization solution containing per 100 mls: 50 mls formamide, 25mls of 20×SSPE (1×SSPE=0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7),2.5 mls of 20% SDS, 1 ml of 10 mg/ml, sheared herring sperm DNA and 21.5mls of distilled H₂O.

The cloned 650 base pair fragment of Example 2 was used as ahybridization probe of the membrane. The fragment was isolated from thepUC18 plasmid by restriction digestion with EcoR I, electrophoresis in1% agarose, excision of the fragment from the gel and recovery of thefragment from the excised agarose. This purified 650 base pair fragmentof DNA was random prime ³²P labeled using the Megaprime labeling systemaccording to the instructions of the manufacturer (AmershamInternational plc, Buckinghamshire, England). The labeled probe wasdenatured by heating to 100° C. for 5 minutes and immediately added tothe prehybridization solution containing the membrane. The hybridizationreaction proceeded for 18 hours at 37° C. with gentle shaking. Themembrane was rinsed in a solution of 2×SSC/0.3% SDS and then washed for15 minutes in the same solution at 37 ° C. with shaking. The membranewas further washed with a solution of 0.2×SSC/0. 1% SDS at 37° C. for 30minutes. The membrane was then exposed onto X-Omat AR film (EastmanKodak Co, Rochester, N.Y.) for 3 hours and developed.

The film developed from the digests prepared as above showed only oneband of hybridization per restriction enzyme digestion consistent withhybridization. The EcoRI digestion showed a single band of hybridizationat about 5.5 kb in length. Because this hybridized fragment was anexcellent candidate fragment to contain a gene corresponding to theentire 38 kD esterase as a result of its size being consistent with agene encoding a protein of that size, a sub-library was made choosingEcoR I to digest genomic Aspergillus niger DNA to obtain fragment sizesaround 5.5 kb in length.

A restriction digest was made on genomic DNA prepared as in Example 3.The reaction included 50 μl of genomic DNA, 50 μl of 10× restrictionendonuclease buffer H (Boehringer Mannheim), 25 μl of EcoRI (10units/μl, Boehringer Mannheim), 375 μl of distilled water. The reactionproceeded at 37° C. for 6 hours. The digestion mixture waselectrophoresed through 0.8% agarose. Fragments between a range ofapproximately 5 kb to 6 kb were cut from the gel in three approximatelyequal slices. The three pools of DNA fragments contained within thethree gel slices each possessed a slightly different range of fragmentlengths. The DNA was recovered from the slices of agarose using Q/AquickGel Extraction columns, following the instructions of the manufacturer(Qiagen, Inc., Chatsworth, Calif.). Approximately {fraction (1/10)} ofeach pool of recovered DNA was electrophoresed in 0.8% agarose andsouthern hybridized to the 650 base pair fragment as described above.The pool of DNA which gave the strongest hybridization signal wasligated into an EcoR I digested E. coli vector (for example pLITMUS 28,New England Biolabs), which was then transformed into E. coli . The E.coli transformants were plated out on 5 plates at a concentration ofapproximately 500 colonies per plate (150 mm diameter plate). Colonylifts were performed on the plates using Maximum Strength Nytran Plusmembranes. A southern hybridization was performed using the 650 basepair fragment. Four strong hybridization signals were obtained. Coloniesputatively corresponding to the four strong hybridization signals weregrown up and their plasmid DNA recovered. Restriction digests on theplasmid DNA were made using restriction enzymes that were chosen basedon sites within the 650 base pair fragment. One plasmid restrictiondigest gave restriction fragments consistent with the known restrictionsites within the 650 base pair fragment. Upon DNA sequencing, this clonewas revealed to contain the 650 base pair sequence that was obtainedthrough PCR described in example 2. Restriction mapping of this clonereveals the 650 base pair fragment to lie within the approximately 5.5kb of cloned genomic DNA sequence. Based on this procedure, DNA encodingthe entire gene of the 38 kD esterase was isolated corresponding to thesequence provided in FIG. 5 (SEQ. ID. NO:27) encoding a protein havingthe amino acid sequence of FIG. 5 (SEQ. ID. NO:28).

Modifications of this method which are known to effect similar resultswould also be effective in obtaining the suitable DNA or clones. Ofcourse, this method is similarly suitable for the identification andcloning of homologous esterase enzymes from species other thanAspergillus niger. For example, as described above, a genomic librarycould be produced from a suitable microorganism by preparing genomic DNAand cutting with an appropriate restriction endonuclease. The librarywould then be subjected to Southern Blot hybridization with the 650 basepair fragment described in Example 2 as a probe and suitable hybridizingfragments ligated into a suitable expression vector and transformed intoa suitable organism for expression. Suitable techniques for suchprocesses are described in, for example, European Patent No. 215 594(Genencor).

Example 5 Construction of an Expression System for FAE

Production of FAE was achieved by constructing an expression vector andtransforming that vector into Aspergillus. The transformed Aspergillusstrain is then grown in appropriate fermentation media. An FAEexpression vector is described below. Transformation of Aspergillus isknown in the art and is described for example in “Cloning, mapping andmolecular analysis of the pyrG (orotidine-5′-phosphate decarboxylase)gene of Aspergillus nidulans”, B. Oakley et al., Gene, 61 (1987) pp.385-399.

An FAE expression vector can be constructed in available E. coliplasmids like pNEB193 (New England Biolabs, Beverly, Mass.). Threeelements are required for the expression vector. In brief these elementsare: The FAE gene with its downstream terminator sequence, the A. nigerglucoamylase promoter and the A. nidulans pyrG gene which is used as aselectable marker for transformation. The pyrG gene may be PCR amplifiedfrom Aspergillus nidulans FGSC4 obtainable from the Fungal GeneticsStock Center, Department of Microbiology, University of Kansas MedicalCenter, Kansas City, Kans. 66160-7420 USA. The FAE gene sequence isgiven in FIG. 6. The A. niger glucoamylase promoter and the A. nidulanspyrG DNA sequences may be obtained from the GenBank sequence database.The A. nidulans pyrG sequence is disclosed in Oakley et al. The DNAsequence of the A. niger glucoamylase promoter is disclosed in“Regulation of the glaA gene of Aspergillus niger,” Fowler et al.,Current Genetics (1990) 18:537-545. The elements were arranged in the E.coli plasmid in such a way that the glucoamylase promoter drives theexpression of the fae1 gene starting from the fae1 start methioninecodon (from base 519 in the fae1 gene). This allows the strongglucoamylase promoter to drive expression of the FAE gene product.

Methods for constructing DNA sequences in E. coli plasmids are known inthe art. An acceptable method for constructing an FAE expression vectorin the vector pNEB193 follows:

(a) PCR is used to amplify the A. nidulans pyrG gene and insert thissequence into pNEB193. This could be accomplished with two primers andsuitable conditions to obtain a pyrG fragment of approximately 2.0 kb insize. For example, the upper primer may be:

5′-GGCCTGCAGCCCCGCAAACTACGGGTACGTCC-3′ (SEQ.ID.NO:30)

and the lower primer may be:

5′-CGCGCTGCAGGCTCTTTCTGGTAATACTATGCTGG-3′ (SEQ.ID.NO:31)

Aspergillus nidulans genomic DNA may be prepared for amplification asdescribed above. The conditions needed to amplify a 2.0 kb fragment areknown in the art, for example they are given in the “Expand HighFidelity PCR System” (Boehringer Mannheim, Indianapolis, Ind.). Afteramplification of the fragment, it is isolated and then digested with theenzyme Pst I. Also the plasmid pNEB193 is digested with Pst I. Afterdigestion, the fragment and plasmid are isolated and ligated together.

(b) PCR is used to amplify the A. niger glucoamylase promoter and placethis sequence into the plasmid constructed. This could be accomplishedusing two primers and conditions to obtain a promoter fragment ofapproximately 1.9 kb in size. As examples of suitable primer, the upperprimer could be

5′-GGCTTAATTAACGTGCTGGTCTCGGATCTTTGGCGG-3′ (SEQ.ID.NO:32)

and the lower primer could be:

5′-GGGGCGCGCCAGATCTAGTACCGATGTTGAGGATGAAGCTC-3′ (SEQ.ID.NO:33).

While many different strains are suitable for amplification, oneparticularly useful strain for amplification is A. niger strainATCC10864 (American Type Culture Collection, Rockville, Md.). The A.niger genomic DNA for amplification may be isolated as described above.After amplification the fragment is isolated and digested with theenzymes Pac II and Asc I. The plasmid created in (a) above above is alsobe digested with the enzymes Pac II and Asc I. The digests of both theamplified fragment and plasmid would be ligated together.

(c) Two fragments of the FAE gene are combined into the plasmid createdin (b) utilizing the a 5.5 kb EcoRI fragment comprising the entire FAEgene. The first fragment is created via PCR using the following primersin connection with the 5.5 kb EcoRI fragment of the FAE gene disclosedabove as the source to be amplified:

forward primer: 5′-GCCCAGATCTCCGCAATGAAGCAATTCTCCGCCAAACAC-3′(SEQ.ID.NO:34)

reverse primer: 5′-AATAGTCGACGGAATGTTGCACAGG-3′ (SEQ.ID.NO:35)

This fragment is digested with Bgl II and Sal I to result in a fragmentof about 169 base pairs long. The second fragment is made by incubatingthe 5.5 kb EcoRI fragment of the FAE gene with SalI and EcoRI, theresulting 1.75 kb fragment being isolated. The plasmid created in (b)above is prepared for insertion of the FAE gene by digesting with Bgl IIand EcoRI. The three fragments, the 169 base pair PCR product, the 1.75kb fragment and the Bgl II/EcoRI digested step 2 plasmid, are ligatedtogether. This resulting plasmid would be an Aspergillus FAE geneexpression vector.

The vector created above would be used to carry out transformation ofAspergillus.

Example 6 Identification of Homologous Genes in Filamentous Fungi

A southern hybridization experiment was performed under hybridizationconditions described using 25% formamide in hybridization buffer asdefined herein. The 650 base pair FAE gene fragment isolated in Example2 was used to probe digested genomic DNA from a number of genera.Hybridization bands were obtained with genomic DNA obtained from fungiother than Aspergillus niger implying the existence of homologousesterase genes in these other organisms. Based on the hybridizationdata, it is believed that the DNA identified in this experiment willcode for closely related enzymes with esterolytic activity. The genesfor these other homologous enzymes are cloned by the methods described.These cloned genes are then expressed in suitable hosts to produce theencoded enzyme.

The genomic DNA was digested with two restriction enzymes, Bgl II andPpu10 I, and then electrophoresed through 0.7% agarose in two differentgels. Genomic DNA fragment sizes separated on the agarose gel rangedfrom about 1 kb to about 20 kb. The gels were depurinated and denaturedand Southern blotted onto Nytran plus. The membranes were air dred andhybridized with the 650 base pair fragment ³²P labeled. The membraneswere washed under low stringency conditions, followed by washing understandard stringency conditions. The membranes were thenautoradiographed. The reproduced gels are provided in FIGS. 6 and 7.

lane # endonuclease DNA source gel 1 2 Bgl II digest Aspergillus nigerGCI strain #7 3 Ppu10 I digest Aspergillus niger GCI strain #7 4 Bgl IIdigest Aspergillus terrus 5 Ppu10 I digest Aspergillus terrus 6 Bgl IIdigest Trichoderma reesei strain QM6a, ATCC13631 7 Ppu10 I digestTrichoderma reesei strain QM6a, ATCC13631 8 Bgl II digest Acremoniumbrachypenium, ATCC 32206 9 Ppu10 I digest Acremonium brachypenium, ATCC32206 gel 2 17  Bgl II digest Aspergillus niger GCI strain #7 18  Ppu10I digest Aspergillus niger GCI strain #7 19  Bgl II digest Gliocladiumroseum 20  Ppu10 I digest Gliocladium roseum 25  Bgl II digestPenicillium notatum 26  Ppu10 I digest Penicillium notatum

Bands are apparent in lanes 2, 3, 17 and 18 which correspond to thecloned FAE gene described in this patent. Bands appear in the lanes 2and 17 Bgl II digest which may indicate other homologous FAE enzymespresent in the Aspergillus niger strain. Two bands are present in lanes4 and 5 and two bands are present in lane 4, indicating homologous DNAin the Aspergillus terrus. A band is apparent in lane 7 indicatinghomologous DNA in Trichodermna reesei. A band is apparent in lane 8indicating homologous DNA in Acremonium brachypenium. A band is apparentin lane 19 indicating homologous Gliocladium roseum. Two bands arepresent in lanes 25 and 26 indicating homologous DNA in Penicilliumnotatum.

Example 7 Biochemical Properties and Substrate Specificity of FAEPurified According to Example 1

The 38 kD esterase isolated according to Example 1 was analyzed forbiochemical properties. The molecular weight was found to be about 38 kDwhen measured on SDS-PAGE and 30-32 kD as measured by HPSEL. The pl asmeasured on isoelectric focusing (IEF) gel was found to be about 2.8.Purified 38 kD esterase was found to be active toward several naturalferuloyl and p-coumaroyl esters, cell walls of wheat bran and sugar beetpulp, wheat flour, the pentosan fraction of wheat flour, and ethyl andmethyl esters of ferulic and p-coumaric acid. Kinetic data for varioussubstrates is presented in Table 1. The 38 kD esterase showed a pHoptimum of 5.1 for methyl ferulate with 83% and 25% maximal activityfound at pH 3 and 8, respectively. When the 38 kD esterase was incubatedin buffer for 30 minutes without substrate at pH 5.1, the temperatureoptima was 55° C. With 250 μM methyl ferulate present the optimaincreases to 65° C. A low K_(m) of the trisaccharide FAXX favors the useof the 38 kD esterase of the present invention in combination with axylanase that leaves such carbohydrate oligomers preferentiallyunhydrolyzed when degrading cell walls.

Purified 38 kD esterase was analyzed for a variety of biochemicalactivities with an API-20 enzyme test strip (BioMerieux Vitek) accordingto the manufacturers instructions. The results shown in Table 2.Activity was observed on the following substrates. “+++” indicates verystrong response, “++” indicates strong response and “+” indicatesactivity shown towards substrate. “−” means no activity detected.

TABLE 1 Activity of 38kD Esterase On Various FeruloylatedOligosaccharides Substrate K_(m) (mM) V_(max) (U/mg) V_(max)/K_(m)Methyl-FA 2.08  87 41.8 FA 0.125 245 1960.0 FAX 0.078 276 3539.5 FAXX0.019 498 26210.5 FAX₃ 0.052 307 5903.8

TABLE 2 Substrate Specificity For 38 kD Esterase Enzyme GenerallyAssociated Substrate Activity With Activity 2-naphthyl butyrate [pH 6.5](+++) Esterase (C4) 2-naphthyl caprylate [pH 7.5] (++) Esterase/Lipase(C8) 2-naphthyl myristate [pH 7.5] (+) Lipase (C14) 2-naphthyl phosphate[pH 5.4] (+) Acid Phosphotase Naphthol-AS-BI-phosphate [pH 5.4] (++)Phosphohydrolase 6-Br-2-naphthyl-αD-galactopyranoside [pH 5.4] (+++)α-galactosidase 2-naphthyl-βD-galactopyranoside [pH 5.4] (++)β-galactosidase 2-naphthyl-αD-glucopyranoside [pH 5.4] (+) α-glucosidase6-Br-2-naphthyl-βD-glucopyranoside [pH 5.4] (+++) β-glucosidase2-naphthyl phosphate [pH 8.5] − Alkaline PhosphotaseL-leucyl-2-naphthylamide [pH 7.5] − Leucine arylamidaseL-valyl-2-naphthylamide [pH 7.5] − Valine arylamidaseL-cystyl-2-naphthylamide [pH 7.5] − Cysteine ArylamidaseN-glutaryl-phenylalanine-2-naphthylamide − ChymotrypsinN-benzoyl-DL-arginine-2-naphthylamide [pH 8.5] − TrypsinNaphthol-AS-BI-βD-glucuronide [pH 5.4] − β-Glucuronidase6-Br-2-naphthyl-αD-mannopyronoside [pH 5.4] − α-Mannosidase2-naphthyl-αL-fucopyranoside [pH 5.4] − α-Fucosidase

Example 8 Activity of the 38 kD Esterase Towards Sugar Beet PulpSubstrate

Sugar beet pulp (100 mg SBP) was incubated together with FAE (1.5 FAXXUnits as measured by the method described in McCallum et al., AnalyticalBiochemistry, vol. 196, p. 362 (1991)) alone and in combination of 50Units xylanase from Trichoderma longibrachiatum (Irgazyme 4×, availablecommercially from Genencor International, Inc.) in sodium acetate buffer(100 mM, pH 5.0). The reaction mixtures were continuously inverted at25° C. during incubation. SBP incubated with (i) buffer alone, (ii)xylanase alone, or (iii) boiled FAE served as controls. Reactions werehalted at 12 and 24 hours by the addition of 1.1 equivalents of HCL.Determination of total ferulic acid content of SBP was determined bysaponification with NaOH by the method of Bomeman et al., Appl.Microbiol. Biotech. vol. 33, pp. 345-351 (1990). Ferulic acid releasedby enzymatic treatment was determined by HPLC using authentic ferulicacid standards (Aldrich) by the method of Bomeman et al., Anal.Biochem., vol. 190, pp.129-133 (1990). Results are shown in table 3.

TABLE 3 Release of ferulic acid from sugar beet pulp with ferulic acidesterase Ferulic acid released from sugar beet pulp Enzyme 12 hrs 24 hrstreatment μg % μg % FAE 15.3 2.7 26.2 4.6 Xylanase 0.5 0.1 0.6 0.1 FAE +27.1 4.8 49.7 8.7 Xylanase Buffer Control 0.2 0.04 0.2 0.04 Inactivated0.2 0.03 0.2 0.04 FAE

Of course, it should be understood that a wide range of changes andmodifications can be made to the preferred embodiment described above.It is therefore intended that the foregoing detailed description beunderstood in the context of the following claims, including allequivalents, which are intended to define the scope of this invention.

35 32 amino acids amino acid single linear 1 Ala Ser Thr Gln Gly Ile SerGlu Asp Leu Tyr Ser Arg Leu Val Glu 1 5 10 15 Met Ala Thr Ile Ser GlnAla Ala Tyr Xaa Asp Leu Leu Asn Ile Pro 20 25 30 8 amino acids aminoacid single linear 2 Xaa Thr Val Gly Phe Gly Pro Tyr 1 5 9 amino acidsamino acid single linear 3 Phe Gly Leu His Leu Xaa Gln Xaa Met 1 5 8amino acids amino acid single linear 4 Xaa Ile Ser Glu Asp Leu Tyr Ser 15 9 amino acids amino acid single linear 5 Tyr Ile Gly Trp Ser Phe TyrAsn Ala 1 5 10 amino acids amino acid single linear 6 Gly Ile Ser GluAsp Leu Tyr Xaa Xaa Gln 1 5 10 10 amino acids amino acid single linear 7Xaa Ile Ser Glu Ser Leu Tyr Xaa Xaa Arg 1 5 10 7 amino acids amino acidsingle linear 8 Gly Ile Ser Glu Asp Leu Tyr 1 5 7 amino acids amino acidsingle linear 9 Leu Glu Pro Pro Tyr Thr Gly 1 5 14 amino acids aminoacid single linear 10 Xaa Ala Asn Asp Gly Ile Pro Asn Leu Pro Pro ValGlu Gln 1 5 10 8 amino acids amino acid single linear 11 Tyr Pro Asp TyrAla Leu Tyr Lys 1 5 22 base pairs nucleic acid single linear 12CGGGAATTCG CWSACCARGG AT 22 32 amino acids amino acid single linear 13Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Ser Arg Leu Val Glu 1 5 1015 Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Leu Asn Ile Pro 20 2530 25 base pairs nucleic acid single linear 14 CGGGAATTCT AYTAYATHGGTGGGT 25 16 amino acids amino acid single linear 15 Val His Gly Gly TyrTyr Ile Gly Trp Val Ser Val Gln Asp Gln Val 1 5 10 15 25 base pairsnucleic acid single linear 16 CGGGAATTCA CCCACCDATR TARTA 25 16 aminoacids amino acid single linear 17 Val His Gly Gly Tyr Tyr Ile Gly TrpVal Ser Val Gln Asp Gln Val 1 5 10 15 23 base pairs nucleic acid singlelinear 18 CGGGAATTCT TGGATCCRTC RTT 23 27 amino acids amino acid singlelinear 19 Thr Asp Ala Phe Gln Ala Ser Ser Pro Asp Thr Thr Gln Tyr PheArg 1 5 10 15 Val Thr His Ala Asn Asp Gly Ile Pro Asn Leu 20 25 24 basepairs nucleic acid single linear 20 CGGGAATTCA TCCRTCRTTG CRTG 24 27amino acids amino acid single linear 21 Thr Asp Ala Phe Gln Ala Ser SerPro Asp Thr Thr Gln Tyr Phe Arg 1 5 10 15 Val Thr His Ala Asn Asp GlyIle Pro Asn Leu 20 25 27 base pairs nucleic acid single linear 22CGGGAATTCG CYTGRAAGCR TCGTCAT 27 28 amino acids amino acid single linear23 Met Thr Asp Ala Phe Gln Ala Ser Ser Pro Asp Thr Thr Gln Tyr Phe 1 510 15 Arg Val Thr His Ala Asn Asp Gly Ile Pro Asn Leu 20 25 28 aminoacids amino acid single linear 24 Met Thr Asp Ala Phe Gln Ala Ser SerPro Asp Thr Thr Gln Tyr Phe 1 5 10 15 Arg Val Thr His Ala Asn Asp GlyIle Pro Asn Leu 20 25 650 base pairs nucleic acid single linear 25GCCTCTACGC AGGGCATCTC CGAAGACCTC TACAGCCGTT TAGTCGAAAT GGCCACTATC 60TCCCAAGCTG CCTACGCCGA CCTGTGCAAC ATTCCGTCGA CTATTATCAA GGGAGAGAAA 120ATTTACAATT CTCAAACTGA CATTAACGGA TGGATCCTCC GCGACGACAG CAGCAAAGAA 180ATAATCACCG TCTTCCGTGG CACTGGTAGT GATACGAATC TACAACTCGA TACTAACTAC 240ACCCTCACGC CTTTCGACAC CCTACCACAA TGCAACGGTT GTGAAGTACA CGGTGGATAT 300TATATTGGAT GGGTCTCCGT CCAGGACCAA GTCGAGTCGC TTGTCAAACA GCAGGTTAGC 360CAGTATCCGG ACTATGCGCT GACTGTGACG GGCCACAGGT ATGCCCTCGT GATTTCTTTC 420AATTAAGTGT ATAATACTCA CTAACTCTAC GATAGTCTCG GAGCGTCCCT GGCAGCACTC 480ACTGCCGCCC AGCTGTCTGC GACATACGAC AACATCCGCC TGTACACCTT CGGCGAACCG 540CGCAGCGGCA ATCAGGCCTT CGCGTCGTAC ATGAACGATG CCTTCCAAGC CTCGAGCCCA 600GATACGACGC AGTATTTCCG GGTCACTCAT GCCAACGACG GCATCCCAAA 650 197 aminoacids amino acid single linear 26 Ala Ser Thr Gln Gly Ile Ser Glu AspLeu Tyr Ser Arg Leu Val Glu 1 5 10 15 Met Ala Thr Ile Ser Gln Ala AlaTyr Ala Asp Leu Cys Asn Ile Pro 20 25 30 Ser Thr Ile Ile Lys Gly Glu LysIle Tyr Asn Ser Gln Thr Asp Ile 35 40 45 Asn Gly Trp Ile Leu Arg Asp AspSer Ser Lys Glu Ile Ile Thr Val 50 55 60 Phe Arg Gly Thr Gly Ser Asp ThrAsn Leu Gln Leu Asp Thr Asn Tyr 65 70 75 80 Thr Leu Thr Pro Phe Asp ThrLeu Pro Gln Cys Asn Gly Cys Glu Val 85 90 95 His Gly Gly Tyr Tyr Ile GlyTrp Val Ser Val Gln Asp Gln Val Glu 100 105 110 Ser Leu Val Lys Gln GlnVal Ser Gln Tyr Pro Asp Tyr Ala Leu Thr 115 120 125 Val Thr Gly His SerLeu Gly Ala Ser Leu Ala Ala Leu Thr Ala Ala 130 135 140 Gln Leu Ser AlaThr Tyr Asp Asn Ile Arg Leu Tyr Thr Phe Gly Glu 145 150 155 160 Pro ArgSer Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe 165 170 175 GlnAla Ser Ser Pro Asp Thr Thr Gln Tyr Phe Arg Val Thr His Ala 180 185 190Asn Asp Gly Ile Pro 195 2436 base pairs nucleic acid single linear 27CCATGGTGGT GTCGATATCG GCAGTAGTCT TTGCCGAAAC GTTGAGGGTT ACAGTGATCT 60GCGTCGGACA TACTTCGGGG AATCTACGGC GGAATATCAA AGTCTTCGGA ATATCCATAT 120TGGGAAAGGA CAGAAGCTCC GGGGTAGTTT GATAGATGAG CTCCGGTGTA TTAAATCGGG 180AGCTGACAGG AGTGAGCGTC ATGTAGACCA TCTAGTAATG TCAGTCGCGC GCAATTTCGC 240ACATGAAACA AGTTGATTTC GGGACCCCAT TGTTACATCT CTCGGCTACA GCTCGAGATG 300TGCCTGCCGA GTATACTTAG AAGCCATGCC AGCGTGTTGT TATACGACCA AAAGTCAGGG 360AATATGAAAC GATCGTCGGA TATTTCTTGT TTTTATCCTA AATTAGTCTT CCAGTGGTTT 420ATTTAAGAGA TAGATCCCTT CACAAACACT CATCCAACGG ACTTCTCATA CCACTCATTG 480ACATAATTTC AAACAGCTCC AGGCGCATTT AGTTCAACAT GAAGCAATTC TCCGCCAAAC 540ACGTCCTCGC AGTTGTGGTG ACTGCAGGGC ACGCCTTAGC AGCCTCTACG CAAGGCATCT 600CCGAAGACCT CTACAGCCGT TTAGTCGAAA TGGCCACTAT CTCCCAAGCT GCCTACGCCG 660ACCTGTGCAA CATTCCGTCG ACTATTATCA AGGGAGAGAA AATTTACAAT TCTCAAACTG 720ACATTAACGG ATGGATCCTC CGCGACGACA GCAGCAAAGA AATAATCACC GTCTTCCGTG 780GCACTGGTAG TGATACGAAT CTACAACTCG ATACTAACTA CACCCTCACG CCTTTCGACA 840CCCTACCACA ATGCAACGGT TGTGAAGTAC ACGGTGGATA TTATATTGGA TGGGTCTCCG 900TCCAGGACCA AGTCGAGTCG CTTGTCAAAC AGCAGGTTAG CCAGTATCCG GACTATGCGC 960TGACTGTGAC GGGCCACAGG TATGCCCTCG TGATTTCTTT CAATTAAGTG TATAATACTC 1020ACTAACTCTA CGATAGTCTC GGAGCGTCCC TGGCAGCACT CACTGCCGCC CAGCTGTCTG 1080CGACATACGA CAACATCCGC CTGTACACCT TCGGCGAACC GCGCAGCGGC AATCAGGCCT 1140TCGCGTCGTA CATGAACGAT GCCTTCCAAG CCTCGAGCCC AGATACGACG CAGTATTTCC 1200GGGTCACTCA TGCCAACGAC GGCATCCCAA ACCTGCCCCC GGTGGAGCAG GGGTACGCCC 1260ATGGCGGTGT AGAGTACTGG AGCGTTGATC CTTACAGCGC CCAGAACACA TTTGTCTGCA 1320CTGGGGATGA AGTGCAGTGC TGTGAGGCCC AGGGCGGACA GGGTGTGAAT AATGCGCACA 1380CGACTTATTT TGGGATGACG AGCGGAGCCT GTACATGGTG ATCAGTCATT TCAGCCTCCC 1440CGAGTGTACC AGGAAAGATG GATGTCCTGG AGAGGGCATG CATGTACGTA TACCCGAAGC 1500ACACTTTTTC GGTAAATCAG GACATGTAAT AAGTTCCTTC CATGAATAGA TATGGTTACC 1560CTCACCATAA GCCTTGAGGT TGCCTTTCTC TTTTGATTGT GAATATATAT TTAAAGTAGA 1620TGACAGATAT CTCTAAACAC CTTATCCGCT TAAACCCATC ATAGATTGTG TCACGTGATA 1680GACCCCTTGA ATGATGAGCG AAATGTATCA GTCCCGTTTA AATCAAACCC TTTCAGCCTA 1740GCACAGTCAG AATACACCAA CCCCATTCTA AGGTAGTACT AAATATGAAT ACAGCCTAAA 1800TGCATCGCTA TATGATCCCA TAAAGAAGCA ACAACCTTTC AGATCTCGTT TTGCGCTGCG 1860AAGAGCTAGC TCTACCATGG TCTCAATTAT GAGTGGAGCG TTTAGTCTCG TTTAAGCCTA 1920GCTATCTTAT AAGGACAACA CATGTACATG GGCTTACTTG TAGAGAGGTA GGATCCCGGG 1980CTTCTTCACA TCTCGAGGAG TTGTCTACAC GTCGCGTCCA TGTCATAAGC CGGTACTCGA 2040CGTTGTCGTG ACCGTGACCC AGACCCCTGT TGATAGCGTT GAGAAGGCCC TATATTTGAA 2100TTTCCAATCT CAGCTTTACG AAGATATGCC CATGGTGGAG GGTTAGTAAA CCGATGATGA 2160TCGTGTGCAG CATGAGATGA GACCGTGGCC AATCCTGTTC AAATGCCAAG ACCCGCCTCC 2220TACCACATGT AAGGCATCCG TCGGCCGCAC GTTGAATTGT GCAAATGCCG AGATCATAAA 2280AGCGGCCACA CTTCCACGTC GGTACTGGAT GGGTTGCGCG TGGCCATACT GTGTTTTCCA 2340TTGCGTGGGT CGTTCGTGTT ACTGCGACGC AGATTCTGTA GGCAAGGCGC AGGGCTCTCT 2400TCTGAGGTAG AAAACACCCC ATATTAATCT GAATTC 2436 281 amino acids amino acidsingle linear 28 Met Lys Gln Phe Ser Ala Lys His Val Leu Ala Val Val ValThr Ala 1 5 10 15 Gly His Ala Leu Ala Ala Ser Thr Gln Gly Ile Ser GluAsp Leu Tyr 20 25 30 Ser Arg Leu Val Glu Met Ala Thr Ile Ser Gln Ala AlaTyr Ala Asp 35 40 45 Leu Cys Asn Ile Pro Ser Thr Ile Ile Lys Gly Glu LysIle Tyr Asn 50 55 60 Ser Gln Thr Asp Ile Asn Gly Trp Ile Leu Arg Asp AspSer Ser Lys 65 70 75 80 Glu Ile Ile Thr Val Phe Arg Gly Thr Gly Ser AspThr Asn Leu Gln 85 90 95 Leu Asp Thr Asn Tyr Thr Leu Thr Pro Phe Asp ThrLeu Pro Gln Cys 100 105 110 Asn Gly Cys Glu Val His Gly Gly Tyr Tyr IleGly Trp Val Ser Val 115 120 125 Gln Asp Gln Val Glu Ser Leu Val Lys GlnGln Val Ser Gln Tyr Pro 130 135 140 Asp Tyr Ala Leu Thr Val Thr Gly HisSer Leu Gly Ala Ser Leu Ala 145 150 155 160 Ala Leu Thr Ala Ala Gln LeuSer Ala Thr Tyr Asp Asn Ile Arg Leu 165 170 175 Tyr Thr Phe Gly Glu ProArg Ser Gly Asn Gln Ala Phe Ala Ser Tyr 180 185 190 Met Asn Asp Ala PheGln Ala Ser Ser Pro Asp Thr Thr Gln Tyr Phe 195 200 205 Arg Val Thr HisAla Asn Asp Gly Ile Pro Asn Leu Pro Pro Val Glu 210 215 220 Gln Gly TyrAla His Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr 225 230 235 240 SerAla Gln Asn Thr Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys 245 250 255Glu Ala Gln Gly Gly Gln Gly Val Asn Asn Ala His Thr Thr Tyr Phe 260 265270 Gly Met Thr Ser Gly Ala Cys Thr Trp 275 280 2436 base pairs nucleicacid single linear 29 CCATGGTGGT GTCGATATCG GCAGTAGTCT TTGCCGAAACGTTGAGGGTT ACAGTGATCT 60 GCGTCGGACA TACTTCGGGG AATCTACGGC GGAATATCAAAGTCTTCGGA ATATCCATAT 120 TGGGAAAGGA CAGAAGCTCC GGGGTAGTTT GATAGATGAGCTCCGGTGTA TTAAATCGGG 180 AGCTGACAGG AGTGAGCGTC ATGTAGACCA TCTAGTAATGTCAGTCGCGC GCAATTTCGC 240 ACATGAAACA AGTTGATTTC GGGACCCCAT TGTTACATCTCTCGGCTACA GCTCGAGATG 300 TGCCTGCCGA GTATACTTAG AAGCCATGCC AGCGTGTTGTTATACGACCA AAAGTCAGGG 360 AATATGAAAC GATCGTCGGA TATTTCTTGT TTTTATCCTAAATTAGTCTT CCAGTGGTTT 420 ATTTAAGAGA TAGATCCCTT CACAAACACT CATCCAACGGACTTCTCATA CCACTCATTG 480 ACATAATTTC AAACAGCTCC AGGCGCATTT AGTTCAACATGAAGCAATTC TCCGCCAAAC 540 ACGTCCTCGC AGTTGTGGTG ACTGCAGGGC ACGCCTTAGCAGCCTCTACG CAAGGCATCT 600 CCGAAGACCT CTACAGCCGT TTAGTCGAAA TGGCCACTATCTCCCAAGCT GCCTACGCCG 660 ACCTGTGCAA CATTCCGTCG ACTATTATCA AGGGAGAGAAAATTTACAAT TCTCAAACTG 720 ACATTAACGG ATGGATCCTC CGCGACGACA GCAGCAAAGAAATAATCACC GTCTTCCGTG 780 GCACTGGTAG TGATACGAAT CTACAACTCG ATACTAACTACACCCTCACG CCTTTCGACA 840 CCCTACCACA ATGCAACGGT TGTGAAGTAC ACGGTGGATATTATATTGGA TGGGTCTCCG 900 TCCAGGACCA AGTCGAGTCG CTTGTCAAAC AGCAGGTTAGCCAGTATCCG GACTATGCGC 960 TGACTGTGAC GGGCCACAGG TATGCCCTCG TGATTTCTTTCAATTAAGTG TATAATACTC 1020 ACTAACTCTA CGATAGTCTC GGAGCGTCCC TGGCAGCACTCACTGCCGCC CAGCTGTCTG 1080 CGACATACGA CAACATCCGC CTGTACACCT TCGGCGAACCGCGCAGCGGC AATCAGGCCT 1140 TCGCGTCGTA CATGAACGAT GCCTTCCAAG CCTCGAGCCCAGATACGACG CAGTATTTCC 1200 GGGTCACTCA TGCCAACGAC GGCATCCCAA ACCTGCCCCCGGTGGAGCAG GGGTACGCCC 1260 ATGGCGGTGT AGAGTACTGG AGCGTTGATC CTTACAGCGCCCAGAACACA TTTGTCTGCA 1320 CTGGGGATGA AGTGCAGTGC TGTGAGGCCC AGGGCGGACAGGGTGTGAAT AATGCGCACA 1380 CGACTTATTT TGGGATGACG AGCGGAGCCT GTACATGGTGATCAGTCATT TCAGCCTCCC 1440 CGAGTGTACC AGGAAAGATG GATGTCCTGG AGAGGGCATGCATGTACGTA TACCCGAAGC 1500 ACACTTTTTC GGTAAATCAG GACATGTAAT AAGTTCCTTCCATGAATAGA TATGGTTACC 1560 CTCACCATAA GCCTTGAGGT TGCCTTTCTC TTTTGATTGTGAATATATAT TTAAAGTAGA 1620 TGACAGATAT CTCTAAACAC CTTATCCGCT TAAACCCATCATAGATTGTG TCACGTGATA 1680 GACCCCTTGA ATGATGAGCG AAATGTATCA GTCCCGTTTAAATCAAACCC TTTCAGCCTA 1740 GCACAGTCAG AATACACCAA CCCCATTCTA AGGTAGTACTAAATATGAAT ACAGCCTAAA 1800 TGCATCGCTA TATGATCCCA TAAAGAAGCA ACAACCTTTCAGATCTCGTT TTGCGCTGCG 1860 AAGAGCTAGC TCTACCATGG TCTCAATTAT GAGTGGAGCGTTTAGTCTCG TTTAAGCCTA 1920 GCTATCTTAT AAGGACAACA CATGTACATG GGCTTACTTGTAGAGAGGTA GGATCCCGGG 1980 CTTCTTCACA TCTCGAGGAG TTGTCTACAC GTCGCGTCCATGTCATAAGC CGGTACTCGA 2040 CGTTGTCGTG ACCGTGACCC AGACCCCTGT TGATAGCGTTGAGAAGGCCC TATATTTGAA 2100 TTTCCAATCT CAGCTTTACG AAGATATGCC CATGGTGGAGGGTTAGTAAA CCGATGATGA 2160 TCGTGTGCAG CATGAGATGA GACCGTGGCC AATCCTGTTCAAATGCCAAG ACCCGCCTCC 2220 TACCACATGT AAGGCATCCG TCGGCCGCAC GTTGAATTGTGCAAATGCCG AGATCATAAA 2280 AGCGGCCACA CTTCCACGTC GGTACTGGAT GGGTTGCGCGTGGCCATACT GTGTTTTCCA 2340 TTGCGTGGGT CGTTCGTGTT ACTGCGACGC AGATTCTGTAGGCAAGGCGC AGGGCTCTCT 2400 TCTGAGGTAG AAAACACCCC ATATTAATCT GAATTC 243632 base pairs nucleic acid single linear 30 GGCCTGCAGC CCCGCAAACTACGGGTACGT CC 32 35 base pairs nucleic acid single linear 31 CGCGCTGCAGGCTCTTTCTG GTAATACTAT GCTGG 35 36 base pairs nucleic acid single linear32 GGCTTAATTA ACGTGCTGGT CTCGGATCTT TGGCGG 36 40 base pairs nucleic acidsingle linear 33 GGGGCGCGCC AGATCTAGTA CCGATGTTGA GGATGAAGCT 40 39 basepairs nucleic acid single linear 34 GCCCAGATCT CCGCAATGAA GCAATTCTCCGCCAAACAC 39 25 base pairs nucleic acid single linear 35 AATAGTCGACGGAATGTTGC ACAGG 25 -- 27 -- GC362-2-PCT

We claim:
 1. An isolated DNA encoding the amino acid according to SEQ IDNO:28.
 2. An isolated DNA capable of hybridizing understandard-stringency conditions with a DNA comprising at least 400nucleotides of the DNA sequence according to SEQ ID NO: 29 and whereinsaid isolated DNA encodes a protein having esterolytic activity whichcleaves the ester linkages of phenolic esters.
 3. An expression vectorcomprising the DNA according to claim
 1. 4. A host cell transformed withthe DNA according to claim
 1. 5. A host cell transformed with theexpression vector according to claim
 3. 6. A DNA which encodes a proteinhaving esterolytic activity which cleaves the ester linkage of phenolicesters having a nucleotide sequence of SEQ ID NO: 29 or a portion of thesequence of SEQ ID NO: 29 wherein said portion comprises at least 400nucleotides.
 7. A method of producing an esterase comprising the stepsof: (a) transforming a suitable microbial host cell with an expressionvector comprising the DNA according to claims 1 or 2; and (b)cultivating said transformed host cell under conditions suitable forsaid host cell to produce said esterase.
 8. The method according toclaim 7 further comprising, (c) separating said produced esterase fromsaid host cells to obtain a purified esterase.
 9. A method of isolatinga DNA which encodes a protein having esterolytic activity which cleavesthe ester linkages of phenolic esters comprising: (a) creating a librarycomprising fragments from a first DNA derived from a plant, animal,fungus, yeast or bacteria; (b) combining said library of said first DNAwith a probe comprising a second DNA under low-stringency to effecthybridization between said fragments in said library of DNA and saidprobe wherein said probe comprises DNA corresponding to SEQ ID NO: 29 ora portion thereof comprising at least 100 nucleotides; and (c)separating the hybridized DNA fragments from the non-hybridizedfragments.
 10. The method according to claim 9, wherein said first DNAis derived from a filamentous fungus.
 11. The method according to claim9, wherein said first DNA is derived from Aspergillus.
 12. The methodaccording to claim 9, wherein said conditions suitable for hybridizationcomprise standard-stringency conditions.
 13. The method according toclaim 9, wherein said probe comprises DNA corresponding to a portion ofSEQ. ID NO:29 comprising at least 400 nucleotides.
 14. DNA isolatedaccording to the method of claim
 9. 15. DNA isolated according to themethod of claim
 11. 16. DNA isolated according to the method of claim12.
 17. DNA isolated according to the method of claim 13.